Recombinant Caenolestes fuliginosus NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

What is the basic structure and function of MT-ND4L?

MT-ND4L (mitochondrially encoded NADH dehydrogenase 4L) is a core subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I). The protein from Caenolestes fuliginosus consists of 98 amino acids with a molecular weight of approximately 10.8 kDa. The amino acid sequence is: MASIYLNLMMAFLLALSGVLIYRSHLMSTLLCLEGMMLSLFIMMTLTISHFQMFSLSMAPLILLVFSACEAGIGLALLVKTSNAHGNDHVQNLNLLQC .

It functions in the transfer of electrons from NADH to the respiratory chain, with ubiquinone believed to be the immediate electron acceptor. As part of Complex I, MT-ND4L participates in creating an unequal electrical charge across the inner mitochondrial membrane, which drives ATP production during oxidative phosphorylation .

How does MT-ND4L integrate into the mitochondrial membrane?

MT-ND4L is a multi-pass transmembrane protein embedded in the inner mitochondrial membrane. Research methodologies to study its integration include:

• Hydropathy plot analysis to identify transmembrane domains

• Protein topology mapping using protease accessibility assays

• Fluorescence resonance energy transfer (FRET) analysis with labeled domains

• Cryo-EM structural studies of Complex I

The highly hydrophobic nature of MT-ND4L (particularly evident in mitochondrially-encoded versions) contributes to its membrane integration and anchoring function in Complex I .

What are the key functional domains of MT-ND4L and their conservation across species?

While the complete functional domain architecture hasn't been fully characterized for C. fuliginosus MT-ND4L specifically, comparative analysis with other species shows several conserved features:

• Highly hydrophobic transmembrane regions

• Conserved amino acid residues involved in proton pumping

• Structural motifs that contribute to the core catalytic function

When comparing sequences across species including Canis lupus (MSMVYINIFLAFILSLMGMLVYRSHLMSSLLCLEGMMLSLFVMMSVTILNNHLTLASMMPI VLLVFAACEAALGLSLLVMVSNTYGTDYVQNLNLLQC) , Macaca hecki (MIPTYMNIMLAFTISLLGMLTYRSHLVASLLCLEGMMMSLFIMATLIASNTHFPLINIMPIILLVFAACEAAVGLALLISISNTYGLDYIHNLNLLQC) , and Distoechurus pennatus (MMPINLNLIMAFSLALIGALVYRSHLMSTLLCLEGMMLSLFIQMALLISHFHMFSMSMAPLILLVFSACEAGLGLALLVKTSSNYGNDYVQNLNLLQC) , there is notable conservation especially in the central region of the protein sequence.

What are the optimal expression systems for recombinant C. fuliginosus MT-ND4L production?

For highly hydrophobic mitochondrial proteins like MT-ND4L, several expression systems can be considered:

• Cell-free expression systems: These have proven effective for transmembrane proteins like MT-ND4L from C. fuliginosus . This approach bypasses potential toxicity to host cells and allows for direct incorporation into membrane-mimetic environments.

• E. coli expression: When using bacterial systems, consider:

  • Using specialized E. coli strains designed for membrane protein expression (C41, C43)

  • Expressing with fusion partners to enhance solubility

  • Codon optimization for E. coli

  • Lower induction temperatures (16-20°C) to slow expression and allow proper folding

• Yeast expression systems: For more complex eukaryotic post-translational modifications.

The tag placement is critical - typically an N-terminal His-tag is preferred as seen in currently available recombinant products .

What purification strategies are most effective for recombinant MT-ND4L?

Purification of highly hydrophobic proteins like MT-ND4L requires specialized approaches:

• Detergent selection: Screen multiple detergents (DDM, LDAO, Fos-choline) for optimal extraction without denaturing the protein.

• Affinity chromatography: Utilizing His-tag affinity as the initial purification step, with careful optimization of imidazole concentration in wash and elution buffers.

• Size exclusion chromatography: As a polishing step to separate monomeric protein from aggregates.

• Buffer optimization: Typically, Tris/PBS-based buffers with 50% glycerol at pH 8.0 provide stability .

A recommended reconstitution protocol: Briefly centrifuge the vial prior to opening, reconstitute in deionized sterile water to 0.1-1.0 mg/mL, and add 5-50% glycerol (final concentration) before aliquoting for long-term storage at -20°C/-80°C .

How can researchers verify proper folding and functionality of recombinant MT-ND4L?

Due to its role in Complex I, assessing MT-ND4L functionality requires several approaches:

• Structural assessment:

  • Circular dichroism (CD) spectroscopy to verify secondary structure

  • Limited proteolysis to assess proper folding

  • Native PAGE to examine oligomeric state

• Functional assays:

  • Reconstitution into liposomes or nanodiscs with other Complex I components

  • NADH:ubiquinone oxidoreductase activity assays

  • Proton pumping assays using pH-sensitive fluorescent dyes

• Interaction studies:

  • Pull-down assays with known Complex I interaction partners

  • Blue native PAGE to assess complex formation

  • Crosslinking studies to identify proximity relationships

How does the sequence of C. fuliginosus MT-ND4L compare with other species, and what are the implications?

Comparative sequence analysis between C. fuliginosus MT-ND4L and other species reveals interesting evolutionary patterns:

• Sequence conservation: While there is variability in the N-terminal regions, the central and C-terminal domains show higher conservation, suggesting functional constraints.

• Hydrophobicity profiles: When comparing with nuclear-encoded homologs (like in Chlamydomonas reinhardtii), mitochondrially-encoded versions typically show higher hydrophobicity .

• Genus-specific features: Species-specific amino acid substitutions may relate to metabolic adaptations or environmental pressures.

A methodological approach for comparative analysis:

• Multiple sequence alignment using MUSCLE or CLUSTALW

• Hydropathy plot comparison using Kyte-Doolittle or similar algorithms

• Evolutionary rate analysis using PAML

• Mapping conserved residues onto available structural models

What approaches can be used to study MT-ND4L mutations and their effects on Complex I function?

To investigate the impact of mutations in MT-ND4L:

• Site-directed mutagenesis: Target specific residues based on disease-associated mutations or conserved regions.

• Functional complementation studies: Express wild-type or mutant MT-ND4L in systems lacking endogenous expression to assess functional rescue.

• Structural modeling: Use homology modeling based on available Complex I structures to predict the impact of mutations.

• In vitro reconstitution: Reconstitute purified wild-type and mutant proteins with other Complex I components to measure activity differences.

• RNA interference approaches: As demonstrated in Chlamydomonas studies, RNAi suppression of ND4L expression can reveal its importance in complex assembly .

The T10663C (Val65Ala) mutation in humans has been associated with Leber hereditary optic neuropathy. This mutation appears to disrupt the normal activity of Complex I in the mitochondrial inner membrane, affecting energy production in cells with high energy demands, particularly retinal ganglion cells .

How can recombinant MT-ND4L be used to study mitochondrial disorders?

Recombinant MT-ND4L provides several experimental approaches for mitochondrial disorder research:

• In vitro modeling of mutations:

  • Generate recombinant proteins with disease-associated mutations

  • Compare biochemical properties with wild-type protein

  • Assess impact on Complex I assembly and function

• Protein-protein interaction studies:

  • Identify altered interactions caused by pathogenic mutations

  • Map interaction domains critical for Complex I assembly

• Drug screening platforms:

  • Develop assays using recombinant protein to screen for compounds that rescue mutant phenotypes

  • Test stabilizers of Complex I assembly

• Structural studies:

  • Use purified recombinant protein for structural determination

  • Investigate conformational changes associated with mutations

What are the methodological considerations when using MT-ND4L as a tool to study Leber hereditary optic neuropathy (LHON)?

LHON research using recombinant MT-ND4L requires specific methodological considerations:

• Mutation modeling: The Val65Ala mutation associated with LHON occurs in a conserved region, suggesting functional importance. Researchers should:

  • Engineer this specific mutation in recombinant constructs

  • Compare multiple parameters (stability, assembly, activity)

  • Consider the effect in different cellular contexts

• Tissue-specific effects: Although MT-ND4L functions in all cells with mitochondria, LHON primarily affects retinal ganglion cells. Research approaches should:

  • Investigate cell-type specific vulnerability factors

  • Examine interaction with retinal ganglion cell-specific proteins

  • Test in relevant cellular models (induced pluripotent stem cell-derived retinal cells)

• Bioenergetic profiling:

  • Measure oxygen consumption rates

  • Assess membrane potential changes

  • Quantify ATP production in wild-type vs. mutant conditions

How can structural biology approaches be applied to MT-ND4L research?

Due to its small size (98 amino acids) and hydrophobic nature, MT-ND4L presents unique challenges for structural biology:

• Cryo-electron microscopy (cryo-EM):

  • Can reveal MT-ND4L position within the larger Complex I structure

  • Requires purification of intact Complex I

  • May identify conformational changes during catalysis

• NMR spectroscopy:

  • Suitable for smaller proteins like MT-ND4L

  • Requires isotopic labeling of recombinant protein

  • Can provide dynamic information about protein movements

• X-ray crystallography:

  • Challenging for membrane proteins but possible with crystallization chaperones

  • May require fusion partners or antibody fragments to aid crystallization

  • Benefits from lipidic cubic phase approaches for membrane proteins

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Can provide information about protein dynamics and solvent accessibility

  • Identifies regions involved in protein-protein interactions

  • Useful for comparing wild-type and mutant conformations

What are the considerations for using recombinant MT-ND4L in reconstitution studies of Complex I?

Reconstitution of functional Complex I using recombinant components represents an advanced research application:

• Component preparation:

  • Express and purify multiple Complex I subunits under native conditions

  • Verify individual component folding and stability

  • Use matched expression systems for compatible post-translational modifications

• Membrane mimetic selection:

  • Nanodiscs provide a controlled lipid environment with defined size

  • Liposomes allow for functional assays monitoring proton pumping

  • Detergent micelles may be simpler but less physiologically relevant

• Assembly protocol optimization:

  • Step-wise addition of components may be necessary

  • Monitor assembly using blue native PAGE

  • Verify formation of sub-complexes during the assembly process

• Functional verification:

  • NADH:ubiquinone oxidoreductase activity measurements

  • Electron paramagnetic resonance (EPR) to detect iron-sulfur cluster incorporation

  • Proton pumping assays to confirm vectorial electron transport

Research in Chlamydomonas has shown that absence of ND4L prevents assembly of the 950-kDa whole Complex I and suppresses enzyme activity, highlighting its essential role in complex formation .

How can researchers address the challenge of studying interactions between nuclear-encoded and mitochondrially-encoded subunits of Complex I?

This represents a frontier research question in mitochondrial biology:

• In organello translation systems:

  • Isolated mitochondria can incorporate exogenous recombinant proteins

  • Allow study of integration with endogenously expressed mitochondrial components

• Split fluorescent protein approaches:

  • Tag MT-ND4L with one half of a split fluorescent protein

  • Tag candidate interaction partners with complementary half

  • Monitor assembly through fluorescence complementation

• Proximity labeling methods:

  • Express MT-ND4L fused to enzymes like BioID or APEX2

  • Identify nearby proteins through biotinylation and mass spectrometry

  • Map the interaction network within the native mitochondrial environment

• Comparative analysis of nuclear transfer events:

  • Some species like Chlamydomonas reinhardtii have transferred ND4L to the nuclear genome

  • Study adaptations that facilitate expression and proper targeting

  • Analyze changes in hydrophobicity and targeting sequences